Glucose and/or fructose transporter naglt1 and its gene

ABSTRACT

The present invention provides a novel glucose and/or fructose transporter involved in renal diabetes, its gene, their variant, and their use. The glucose and/or fructose transporter of the present invention and its gene are a glucose and/or fructose transporter involved in renal diabetes and its gene, the protein and the gene are highly expressed in the kidney, and comprise a novel protein that make a larger contribution to the renal glucose reabsorption and its gene. The present invention comprises a variant of the protein and the gene, and an antibody that specifically binds to the protein. Further, the present invention comprises a non-human animal model wherein the gene of the present invention is deficient in its chromosome, a method for screening a preventive/therapeutic drug for renal diabetes, a method for diagnosing glucose and/or fructose transporter function and renal diseases using the gene or the antibody, and a method for regulating a transporter function in a tissue cell by introducing the gene of the present invention into the tissue cell.

TECHNICAL FIELD

The present invention relates to a glucose and/or fructose transporter,its gene, its variant, and their use.

BACKGROUND ART

Although the weight of the human kidney, even in the total weight of twokidneys, is only 0.5% of the total body weight, about 20% of cardiacoutput, that is, 1 to 1.2 L of blood flows into the kidney every minute.About 20% of the blood plasma flow, in other words, 120 mL per minute(200 L per day) is filtered by the glomerulus and will be primary urine.99% of the primary urine is reabsorbed in the renal tubule, and thedaily urine output is the rest of the primary urine, which is 1.5 to 2L. The kidney is constituted of nephrons, the functional unit, andvascular systems surrounding them. The nephron begins at the glomerulus,and reaches to the collecting duct system through the proximal tubule,the intermediate tubular region (thin limb of Henle's loop), and thedistal tubule. Each segment has different function and configuration,and is involved in the concentration and the production of urinecooperatively. Particularly, in cells constituting the proximal tubule,the surface area is large due to the development of the brush bordermembrane of luminal side, and this is an efficient configuration forreabsorbing low-molecular nutritional substances as well as water andelectrolytes in primary urine, and for secreting pharmaceuticals andforeign bodies in blood (The Pharmaceuticals Monthly, Vol. 43, No. 3, pp29-34, 2001; The Pharmaceuticals Monthly, Vol. 42, No. 4, pp 113-120,2000).

In the basolateral membrane of blood side and the brush border membraneof luminal side in epithelial cells of the proximal tubule, transporterswhich mediate the reabsorption and the secretion of various substancesthus described are localized, and a network capable ofdirection-selective transportation of substances is formed (BIO Clinica,11, 22-25, 1996; Seitai no Kagaku, 50,268-273, 1999). In the pastseveral years, the cloning and the structural/functional analysis of aglucose transporter gene which regulates renal glucose excretion hasbeen markedly advanced, and a mechanism in the kidney wherein glucosewhich has been transferred into urine in the glomerulus is reabsorbedand returned into blood is being elucidated at molecular level.

In other words, glucose in blood is filtered in the renal glomerulus,and reabsorbed in the renal tubule. By this mechanism, 100% of glucoseis reabsorbed and returned into circulating blood when blood glucoselevel is normal.

Because water-soluble molecules such as glucose and amino acids, andions cannot pass through biomembranes comprising a phospholipid bilayerquickly, transporter proteins which specifically transport thesemolecules generally exist in cell membranes and membrane organelles. Amembrane protein which transports substances intracellulary andextracellulary is a transporter, and such transporters are involved inthe process of transportation of pharmaceuticals, nutrients, etc., whichhas been taken into living bodies, to each tissue, and the excretion ofwastes accumulated in each tissue (Japanese Laid-Open Patent ApplicationNo. 2002-171980). In the kidney, transporters (transport proteins) whichmediate the reabsorption and secretion of various substances arelocalized on the basolateral membrane of blood side and the brush bordermembrane of luminal side, in epithelial cells of the proximal tubule,and a network capable of direction-selective transportation ofsubstances, which takes advantage of environmental characteristics suchas the difference in membrane potentials or pH gradient generatedbetween inside and outside of cells, is formed (BIO Clinica, 11, 22-25,1996; Seitai no Kagaku, 50, 268-273, 1999).

Glucose transporters of vertebrates are roughly classified into twotypes. One is called the facilitated glucose transporter: GLUT, and is atransport carrier of facilitate diffusion type which transportsaccording to glucose concentration gradient generated between inside andoutside of cells. Further, there are eight isoforms for this protein,the 12-transmembrane protein whose molecular weight is about 50,000(Annu. Rev. Physiol. 55, 591-608, 1993; TIBS 23, 476-481, 1998; JapaneseLaid-Open Patent Application No. 2002-218981). Basically, all cellsexpress at least one type of GLUT, thereby obtaining necessary glucoseextracellularly. The other is called the Na⁺-glucose cotransporter:SGLT, an active transport carrier which transports glucose against theconcentration gradient by conjugating Na ion, and is a 14-transmembraneprotein whose molecular weight is about 75,000.

This protein is present on the apical membrane of epithelial cells facedto the luminal side of the small intestine and the kidney, and conductsactive transport wherein glucose is taken into cells with the use ofgradient of electrochemical potential of Na⁺ generated between insideand outside of cells (Physiol. Rev. 74, 993-1026, 1994; Am. J. Physiol.276, (5 Pt 1), G1251-1259, 1999; Japanese Laid-Open Patent ApplicationNo. 2002-218981). Glucose and Na⁺ taken into epithelial cells by SGLTare released into blood by the action of Na⁺—K⁺ pump and GLUT-2 presenton the basolateral membrane.

SGLTs of mammals are further classified into two types, SGLT1 and SGLT2,according to their transport property. SGLT1 transports two Na⁺ ions perone molecule of sugar, has high affinity to glucose and galactose, andis expressed in the small intestine and the kidney. On the other hand,SGLT2 transports one Na⁺ ion per one molecule of sugar, has low affinityto glucose, and does not transport galactose. This protein is expressedin the kidney, however, its expression is not observed in the smallintestine. In addition, SGLT1 and SGLT2 function in different sites inthe kidney. Most of glucose transferred into urine in the glomerulus isfirst reabsorbed by SGLT2 in the proximal tubule, and further,completely reabsorbed by SGLT1 in the distal tubule, and returned intoblood. By contrast, all glucose in food is absorbed into the body bySGLT1 in the small intestine.

As mentioned above, glucose transporters SGLT1 and SGLT2 are expressedin the kidney, and it is considered that the reabsorption process ofglucose, which has been filtered in the glomerulus, into epithelialcells of renal tubules is mediated by these two transporters.

On the other hand, there is a condition called renal diabetes, whereinglycosuria is clearly observed even though glucose concentration inplasma is in the normal range (170 mg/dL or less). It is most often thecase that the maximum rate (shown by TmG) that glucose which has beenpassed through the glomerulus is reabsorbed in the renal tubule, whichis 350 mg/min for normal persons, is abnormally low. As a result, theglucose concentration in urine becomes abnormally high irrespective ofthe glucose concentration in plasma. This phenomenon is also observed incases of abnormal proximal tubule, in other words, congenital oracquired Fanconi syndrome, or after phloridzin injections. As anothercause of renal diabetes, there is a case wherein apparent threshold ofglucose is lowered but both mean value of threshold and Tmg areperfectly normal. Abnormal increase in the excretion of glucose intourine found in such case is found only when the glucose concentration inplasma is low. Glucose excretion at plasma level exceeding the maximumthreshold is rather normal (Medical Dictionary, 18^(th) Ed., pp1059-1060, Nanzando Co., Ltd., 1998; Biochemical Dictionary, 2^(nd) Ed.,pp 673, Tokyo Kagaku Dozin Co., Ltd., 1990).

Renal diabetes is thought to be caused by the a defect in renal glucosereabsorption, and the defect in glucose reabsorption is thought to becaused by congenital or acquired deficiency of known two types oftransporters, in other words, SGLT1 and SGLT2, which are considered tomediate reabsorption process of glucose filtered in the glomerulus intoepithelial cells of the renal tubule in the kidney. However, its causalgene has not been identified yet, and it has been considered to becaused by congenital or acquired deficiency of the known SGLT1 and SGLT2genes, and other unknown transporter genes.

The development of pharmaceuticals for targeting or avoiding renaldiseases is important to conduct drug therapy for renal diseases moreefficiently and safely, however, there is no efficient successfulexamples in the present circumstances due to the lack of the pasttechnical study basis for elucidating the cause of renal diseases.

The object of the present invention is to provide a novel glucosetransporter which is expressed in the kidney and involved in renalglucose reabsorption, its gene, their variants, and their use.

It is thought that renal diabetes is caused by a defect in renal glucosereabsorption, and that the defect is caused by congenital or acquireddeficiency of known glucose transporter genes, SGLT1 and SGLT2, however,its causal gene has not been identified yet. The present inventors haveconducted intensive search for the unidentified gene, and have found agene, other than the known SGLT1 and SGLT2 glucose transporter genes,which is highly expressed in the kidney, and the present invention hasbeen completed.

The glucose transporter of the present invention shows higher amount ofexpression than that of the known SGLT1 and SGLT2, and makes a largercontribution to the renal glucose reabsorption, while it has a propertyto recognize glucose specifically. The glucose transporter of thepresent invention is designated as “glucose transporter NaGLT1”. Inaddition, as a result of study, it is found that NaGLT1 of the presentinvention has Na⁺-dependent fructose transporter function.

The present invention also comprises a glucose and/or fructosetransporter NaGLT1 and a variant of its gene, and an antibody thatspecifically binds to the glucose and/or fructose transporter NaGLT1.The present invention further comprises a non-human animal model whichdevelops renal diabetes, whose gene function to express a polypeptidewhich has glucose and/or fructose transporter function of the presentinvention is deficient in its chromosome, and the screening of apreventive/therapeutic drug for renal diabetes with the use of thenon-human animal model, and moreover, a method for diagnosing glucoseand/or fructose transporter function and renal diseases with the use ofthe gene and the antibody of the present invention and a diagnostic drugfor the method. The present invention still further comprises a methodfor regulating glucose and/or fructose transporter function in an animaltissue cell by controlling the expression of the gene of the presentinvention with the use of an antisense strand DNA, etc.

DISCLOSURE OF THE INVENTION

The present invention specifically comprise; a DNA which comprises abase sequence shown by SEQ ID NO: 1 in the sequence listing or itscomplementary sequence, or a sequence containing part or whole of thesesequences (“1”), a DNA which hybridizes with the DNA according to “1”under a stringent condition, and which encodes a polypeptide havingglucose and/or fructose transporter function (“2”), a DNA which encodesthe following polypeptide (a) or (b); (a) a polypeptide which comprisesan amino acid sequence shown by SEQ ID NO: 2 in the sequence listing,(b) a polypeptide which comprises an amino acid sequence wherein one ora few amino acids are deleted, substituted or added in the amino acidsequence shown by SEQ ID NO: 2 in the sequence listing, and which hasglucose and/or fructose transporter function (“3”), a polypeptide whichcomprises an amino acid sequence shown by SEQ ID NO: 2 in the sequencelisting (“4”), a polypeptide which comprises an amino acid sequencewherein one or a few amino acids are deleted, substituted or added inthe amino acid sequence shown by SEQ ID NO: 2 in the sequence listing,and which has glucose and/or fructose transporter function (“5”), amethod for producing a polypeptide which has glucose and/or fructosetransporter function, wherein the DNA according to “1” to “3” isincorporated into an expression vector and expressed by introducing therecombinant expression vector into a host cell (“6”), an antibody whichis induced by using the polypeptide according to “4” or “5”, and whichbinds to the polypeptide specifically (“7”), the antibody according to“7”, wherein the antibody is a monoclonal antibody (“8”), the antibodyaccording to “7”, wherein the antibody is a polyclonal antibody (“9”)

The present invention also comprises; a method for producing an animaltissue cell expressing a polypeptide which has glucose and/or fructosetransporter function, wherein the DNA according to any one of “1” to “3”is introduced into an animal tissue cell (“10”), the method forproducing an animal tissue cell expressing a polypeptide which hasglucose and/or fructose transporter function according to “10”, whereinthe animal tissue cell is a tissue cell of rat kidney, an epithelialcell derived from porcine kidney, an epithelial cell derived from caninekidney or an epithelial cell derived from opossum kidney (“11”), themethod for producing an animal tissue cell expressing a polypeptidewhich has glucose and/or fructose transporter function according to“10”, wherein the animal tissue cell is REK293, a transfected humanembryonic kidney cell line (“12”), an animal tissue cell expressing apolypeptide which has glucose and/or fructose transporter function,which is produced by the method according to any one of “10” to “12”(“13”), a method for screening a substance having a glucose and/orfructose transporter function-regulating activity, wherein an effect ofa test substance on glucose transport function is measured with the useof the animal tissue cell expressing a polypeptide which has glucoseand/or fructose transporter function according to “13” (“14”), anon-human animal model which develops renal diabetes caused by a defectin renal glucose reabsorption, whose gene function to express apolypeptide which has glucose and/or fructose transporter function shownby SEQ ID NO: 2 in the sequence listing is deficient in its chromosome(“15”), the non-human animal model which develops renal diabetesaccording to “15”, wherein the deficiency in the gene function toexpress a polypeptide which has glucose and/or fructose transporterfunction is deficiency in a function of a gene which expresses apolypeptide which has glucose and/or fructose transporter function shownby SEQ ID NO: 1 in the sequence listing (“16”), a method for screening apreventive/therapeutic drug for renal diabetes caused by a defect inglucose reabsorption, wherein a test substance is administered to thenon-human animal model which develops renal diabetes caused by a defectin renal glucose and/or fructose reabsorption according to “15” or “16”,and glucose reabsorption ability of the non-human animal model, or acell, a tissue or an organ of the non-human animal model ismeasured/evaluated (“17”), a probe for diagnosing glucose and/orfructose transporter function comprising whole or part of an antisensestrand of the base sequence according to “1” (“18”), a microarray or aDNA chip for diagnosing glucose and/or fructose transporter function,wherein at least one DNA according to any one of “1” to “3” isimmobilized (“19”).

The present invention further comprises; a pharmaceutical for diagnosingglucose and/or fructose transporter function, wherein the antibodyaccording to any one of “7” to “9” and/or the probe for diagnosingaccording to “18” is prepared (“20”), a method for diagnosing glucoseand/or fructose transporter function, wherein a sample is obtained froma test substance, and the expression of the gene according to “1” in thesample is measured (“21”), a method for diagnosing glucose and/orfructose transporter function, wherein the measurement of the geneexpression according to “21” is conducted with the probe for diagnosingglucose and/or fructose transporter function according to “18”, or withthe microarray or the DNA chip for diagnosing glucose and/or fructosetransporter function according to “19” (“22”), a method for diagnosingglucose and/or fructose transporter function, wherein a sample isobtained from a test substance and cultured, and the polypeptideaccording to “4” produced by the expression of the gene in the sample ismeasured (“23”), a method for diagnosing glucose and/or fructosetransporter function, wherein the measurement of the polypeptideaccording to “23” is conducted with the antibody according to any one of“7” to “9” (“24”), a method for diagnosing a renal disease, wherein thediagnosis of glucose and/or fructose transporter function according toany one of “21” to “24” is measurement of glucose and/or fructosetransporter function in a renal disease (“25”), a method for regulatingglucose and/or fructose transporter function in an animal tissue cell,wherein the DNA according to any one of “1” to “3” is introduced into ananimal tissue cell (“26”), a method for regulating glucose and/orfructose transporter function in an animal tissue cell, wherein theexpression of the DNA according to “1” is suppressed in an animal tissuecell (“27”), a method for regulating glucose and/or fructose transporterfunction in an animal tissue cell, wherein the expression of the DNAaccording to “1” is suppressed in an animal tissue cell by introducingwhole or part of an antisense strand of the DNA base sequence accordingto “1” into an animal tissue cell (“28”), the method for regulatingglucose and/or fructose transporter function in an animal tissue cellaccording to any one of “26” to “28”, wherein the animal tissue cell isan animal kidney cell (“29”).

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: (A) shows the base and amino acid sequences of NaGLT1 of thepresent invention, and (B) shows hydropathy plot (hydrophobicityanalysis) of the amino acid sequence of NaGLT1 of the present invention,in Example of the present invention.

FIG. 2: (A) is a photograph showing Northern blot analysis of NaGLT1mRNA in rat cells of the present invention, and (B) is a photographshowing that NaGLT1 mRNA in rat cells of the present invention wasdetected by PCR, in Example of the present invention.

FIG. 3 is a photograph showing renal expression distribution of eachmRNA of NaGLT1, SGLT1, SGLT2, and GAPDH of the present invention,detected by PCR, in Example of the present invention. In the figure, (+)shows the case wherein RT-PCR was conducted under the condition thatreverse transcriptase was contained, and (−) shows the case whereinRT-PCR was conducted under the condition that reverse transcriptase wasnot contained, respectively.

FIG. 4 shows that the expression amount of NaGLT1, SGLT1, and SGLT2 mRNAof the present invention was analyzed quantitatively by real-time PCR,in Example of the present invention.

FIG. 5 shows the accumulation of various saccharides in oocytes intowhich NaGLT1 mRNA of the present invention which had been synthesized invitro was injected, and in water-injected oocytes, in Example of thepresent invention.

FIG. 6: (A) shows the interdependence between accumulation amount of[¹⁴C]αMeGlc and αMeGlc itself in NaGLT1-expressing oocytes of thepresent invention, (B) shows Hill plot using the data of (A), (C) showsthe interdependence between accumulation amount of [¹⁴C]αMeGlc andextracellular Na⁺ ion concentration, and (D) shows Hill plot using thedata of (C), in Example of the present invention.

FIG. 7 shows the effect of various saccharides on accumulation of[¹⁴C]αMeGlc in NaGLT1-expressing oocytes and water-injected oocytes ofthe present invention, in Example of the present invention.

FIG. 8 is a photograph showing the result of immunoblotting with whichintracellular localization of NaGLT1 protein in rat renal membrane sites(renal crude membrane, brush border membrane, and basolateral membrane)was analyzed in Example of the present invention.

FIG. 9 is a graph showing the uptake of fructose by HEK293 cellsmediated by NaGLT1 in Example of the present invention, and (A) showsthe case of HEK293 cells incubated with buffer solution containingglucose analogs for 15 minutes at 37° C., and (B) shows the uptake of[¹⁴C]fructose by HEK293 cells transfected with NaGLT1 cDNA.

FIG. 10 is a graph showing the uptake of fructose by renal brush bordermembrane vesicles in Example of the present invention, and (A) shows theuptake in the case where membrane vesicles suspended in mannitol andHEPES were incubated with a substrate mixture containing mannitol andHEPES at 25° C., and (B) shows the Na⁺-dependent uptake of fructose inincubation buffer solution at various concentrations.

BEST MODE OF CARRYING OUT THE INVENTION

(The Gene of the Present Invention, a Polypeptide Encoded by the Geneand its Antibody)

The present invention comprises a glucose and/or fructose transporterNaGLT1 involved in the cause of renal diabetes, its causal gene, andtheir variants.

The glucose and/or fructose transporter NaGLT1 cDNA involved in thecause of renal diabetes of the present invention has a base sequenceshown by SEQ ID NO: 1 in the sequence listing. In addition, the glucoseand/or fructose transporter NaGLT1 protein encoded by the cDNA comprisesa polypeptide comprising amino acid sequence shown by SEQ ID NO: 2 inthe sequence listing. The cDNA of glucose and/or fructose transporterNaGLT1, a novel gene obtained in the present invention, comprises 2,173base pairs, and a polypeptide comprising 484 amino acid residues isencoded in its translation region (111^(th) to 1562^(nd) position).

It is confirmed that the glucose and/or fructose transporter NaGLT1 ofthe present invention is an 11-transmembrane glycoprotein according tothe result of hydrophobicity analysis. The glucose and/or fructosetransporter NaGLT1 of the present invention is a glucose and/or fructosetransporter which shows high expression in the kidney, and recognizesnot other monosaccharides but glucose and/or fructose specifically.Expression amount of the glucose and/or fructose transporter NaGLT1 inkidney is higher than that of known glucose transporters SGLT1 or SGLT2,making a great contribution to the renal glucose reabsorption, which isthought to be the cause of renal diabetes.

The gene of the present invention includes: the aforementioned basesequence shown by SEQ ID NO: 1 in the sequence listing or itscomplementary sequence, or a sequence containing part or whole of thesesequences, further, a DNA sequence which hybridizes the base sequenceunder a stringent condition and which encodes a polypeptide havingglucose and/or fructose transporter function, and a DNA which encodesthe following polypeptide (a) or (b);

(a) a polypeptide which comprises an amino acid sequence shown by SEQ IDNO: 2 in the sequence listing,

(b) a polypeptide which comprises an amino acid sequence wherein one ora few amino acids are deleted, substituted or added in the amino acidsequence shown by SEQ ID NO: 2 in the sequence listing and which has apolypeptide having glucose and/or fructose transporter function.

In the present invention, various DNA sequence mutations can beconducted by known gene-mutating means of genetic engineering.

As to the base sequence of the present invention mentioned above, thecondition, “to hybridize the base sequence under a stringent condition”,is exemplified by hybridization at 42° C., and washing treatment at 42°C. with buffer solution containing 1×SSC, 0.1% SDS, and more preferablyexemplified by hybridization at 65° C., and washing treatment at 65° C.with buffer solution containing 0.1×SSC, 0.1% SDS. There are variousfactors other than the temperature condition mentioned above that affectthe hybridization stringency and those skilled in the art can actualizethe same stringency as that of the hybridization referred to in theabove by appropriately combining various factors.

Further, the present invention includes a polypeptide which comprises anamino acid sequence shown by SEQ ID NO: 2 in the sequence listing, apolypeptide which comprises an amino acid sequence wherein one or a fewamino acids are deleted, substituted or added in the amino acid sequenceshown by SEQ ID NO in the sequence listing and which has glucose and/orfructose transporter function. The polypeptide of the present inventioncan be obtained by known technology of genetic engineering. In otherwords, the polypeptide can be obtained by incorporating the gene of thepresent invention into a known expression vector appropriately,introducing the recombinant vector into a host cell, and expressing it.

(Use of Antibodies Induced by the Polypeptide of the Present Invention)

The present invention further includes an antibody induced by thepolypeptide of the present invention and binding to the polypeptidespecifically. As the antibody, a monoclonal antibody and a polyclonalantibody can be exemplified. The antibody can be produced by an ordinarymethod using the polypeptide of the present invention as an antigen. Theantibody of the present invention can be used for detecting theexpression of the gene of the present invention in renal tissue cells,etc., by an antigen-antibody reaction with the polypeptide of thepresent invention which has glucose and/or fructose transporterfunction, and for diagnosing renal diseases relating to the gene. Asimmunoassay using the antibody of the present invention, for example,known immunoassay such as RIA method, ELISA method and fluorescentantibody technique can be used.

(Use of Human Tissue Cells into which the DNA of the Present Inventionis Introduced)

Human tissue cells expressing a polypeptide which has glucose and/orfructose transporter function can be produced by introducing the DNA ofthe present invention into human tissue cells. As for the human tissuecells, it is preferable to use renal tissue cells originally expressingNaGLT1 strongly, and a specific example of the renal tissue cells isHEK293, a transfected human embryonic kidney cell line. In addition, asan animal tissue cell into which the gene of the present invention isintroduced, any of the followings can be also used: a rat kidney tissuecell; an epithelial cell derived from porcine kidney, LLC-PK₁; anepithelial cell derived from canine kidney, MDCK; an epithelial cellderived from opossum kidney, OK. In order to introduce the DNA of thepresent invention in to human tissue cells, appropriate geneintroduction methods such as transfection can be used.

(Use of the Gene of the Present Invention and a Polypeptide Encoded bythe Gene)

In the present invention, a novel gene of NaGLT1, cloned from humanrenal tissues, can be used as a diagnostic probe for diagnosing glucoseand/or fructose transporter function in renal tissue cells with the useof an antisense strand of the base sequence. Further, the gene can beused as a diagnostic drug for glucose and/or fructose transporterfunction with the use of the diagnostic probe and the antibody whichspecifically binds to the polypeptide of the present invention, and as adiagnostic kit containing the diagnostic drug.

In addition, at lease one DNA of the present invention can be fixed on adevice and used as a microarray or a DNA chip for diagnosing glucoseand/or fructose transporter function. Onto the microarray or the DNAchip, other genes of a glucose and/or fructose transporter, etc., can befixed together, and used for diagnosis. Further, in order to measure anexpression status of the gene of the present invention in renal tissuecells, known gene measurement methods such as RT-PCR and Northernblotting can be appropriately used. Genetic diseases in the human kidneycan be detected by measuring the presence or the intensity of expressionof the glucose and/or fructose transporter NaGLT1 gene in renal tissuecells, with the use of the diagnostic method of the present invention.

(Regulation of Glucose and/or Fructose Transporter Function by Using theGene of the Present Invention)

Glucose and/or fructose transporter function in human tissue cells canbe regulated by controlling the expression of the gene of the presentinvention in human tissue cells. The gene expression in human tissuecells can be controlled by introducing the gene of the presentinvention, or an antisense strand for the gene of the present inventioninto human tissue cells to suppress the expression of the gene of thepresent invention. As a method for introducing the gene of the presentinvention into human tissue cells, known gene introduction methods suchas transfection can be used. In order to introduce an antisense strandinto human tissue cells, methods usually used in this field can be used.For example, it is possible to administer an antisense oligonucleotidedirectly into human tissue cells such as renal tissue. In addition, ifnecessary, it is possible to administer together with pharmaceuticallyacceptable intracellular introduction reagents, for instance, lipofectinreagent, lipofectamine reagent, DOTAP reagent, Tfx reagent, liposome andpolymeric carriers, etc.

Genetic diseases in the kidney, etc., can be prevented/treated byregulating glucose and/or fructose transporter function in human tissuecells such as the kidney.

(Gene-Deficient Non-Human Animal Model of the Present Invention and itsUse)

The non-human animal model which develops renal diabetes of the presentinvention means a non-human animal which develops renal diabetes such asa defect in renal glucose and/or fructose reabsorption caused bydeficiency in the function of the glucose and/or fructose transporterNaGLT1 gene in its chromosome. Specific examples of the non-human animalof the present invention include rodents such as a rat, a mouse, aguinea pig, and as a non-human animal model used for the screening ofpreventive/therapeutic drug for renal diabetes of the present invention,a mouse and a rat can be used particularly advantageously, but suchanimals is not limited thereto.

The non-human animal model of the present invention whose function ofNaGLT1 gene is deficient in its chromosome can be produced by knownmethods for producing non-human animal models whose gene is deficient.The method for producing non-human animal models whose function ofNaGLT1 gene is deficient in its chromosome is described below withreference to a mouse whose function of NaGLT1 gene is deficient in itschromosome as an example.

A mouse whose function of NaGLT1 gene is deficient in its chromosome, inother words, a homozygous mutant mouse (−/−) can be constructed, forexample, by the method comprising the steps of: a NaGLT1 gene isscreened by using a gene fragment obtained from rat gene libraryconstructed from rat kidney; part or whole of the screened NaGLT1 geneis substituted with a marker gene, for example, a lac-Z gene, aneomycin-resistant gene or the like, and if necessary, a gene such as adiphthelia toxin A fragment (DT-A) gene or a herpes simplex virusthymidine kinase (HSV-tk) gene is introduced into 5′-terminal side, toconstruct a targeting vector; this constructed targeting vector islinearized and introduced into an ES cell by electroporation or othersuch methods and then homologous recombination is conducted; among thehomologous recombinants, an ES cell resistant to X-gal staining orantibiotics such as G418, ganciclovir (GANC) is selected. It ispreferable to confirm whether the selected ES cell is the intendedrecombinant by Southern blotting or other such methods.

The above-mentioned recombined ES cell is microinjected into a mouseblastocyst, and then the blastocyst is transplanted into a recipientmouse to generate a chimeric mouse. A heterozygous mutant mouse (+/−)can be obtained by intercrossing the chimeric mouse and a wild-typemouse, and a homozygous mutant mouse (−/−) can be obtained byintercrossing the heterozygous mutant mice. As examples of the methodfor confirming whether NaGLT1 gene is deficient in the homozygous mutantmouse include Western blotting or other such methods with which theexpression of NaGLT1 gene in the mouse is confirmed.

The screening of a preventive/therapeutic drug for renal diabetes causedby a defect in renal glucose reabsorption of the present invention isconducted by administering a test substance to the non-human animalwhich develops renal diabetes such as a defect in renal glucose and/orfructose reabsorption caused by deficiency in the function of theglucose and/or fructose transporter NaGLT1 gene in its chromosome, andby measuring/evaluating glucose and/or fructose reabsorption ability ofthe non-human animal model, or a cell, a tissue or an organ of thenon-human animal model.

As mentioned above, when screening a preventive/therapeutic drug forrenal diabetes caused by a defect in renal glucose and/or fructosereabsorption, it is preferable to compare/evaluate with the case of thenon-human animal which develops renal diabetes caused by a defect inglucose and/or fructose reabsorption with the use of a wild-typenon-human animal and/or a non-human animal whose function of NaGLT1 geneis deficient in its chromosome. The wild-type non-human animal means awild-type non-human animal which is the same species as the non-humananimal whose function of NaGLT1 gene is deficient in its chromosome, andthe littermate thereof is more preferably exemplified. The non-humananimal whose function of NaGLT1 gene is deficient in its chromosome canbe produced by the method described previously (Proc. Natl. Acad. Sci.USA 97, 6132-6137, 2000), etc. It is preferable to use a type whosefunction of NaGLT1 gene is deficient and its wild-type littermatesimultaneously because precise comparative experiments, evaluation(analysis) etc., can be conducted at an individual level.

(Regulation of Glucose and/or Fructose Transporter Function Using theGene of the Present Invention)

Glucose and/or fructose transporter function in animal tissue cells canbe regulated by controlling the expression of the gene of the presentinvention in animal tissue cells, in particular, renal tissue cells. Thegene expression in animal tissue cells can be controlled by introducingthe gene of the present invention, or an antisense strand and siRNA forthe gene of the present invention into animal tissue cells to suppressthe expression of the gene of the present invention. As an introductionmethod of the gene of the present invention into animal tissue cells,known gene introduction method such as transfection can be used. Inorder to introduce antisense strands into animal tissue cells, methodsusually used in this field can be used. For example, it is possible toadminister an antisense oligonucleotide directly into animal tissuecells such as renal tissue. In addition, if necessary, it is possible toadminister together with pharmaceutically acceptable intracellularintroduction reagents, for instance, lipofectin reagent, lipofectaminereagent, DOTAP reagent, Tfx reagent, liposome and polymeric carrier,etc.

Genetic diseases in renal diabetes, etc., can be prevented/treated byregulating glucose and/or fructose transporter function in animal tissuecells such as kidney.

The present invention is described below more specifically withreference to Examples, however, the technical scope of the presentinvention is not limited to these exemplification.

EXAMPLE

(cDNA Cloning of the Glucose and/or Fructose Transporter NaGLT1)

Total RNA was extracted from rat kidney by cesium chloridedensity-gradient centrifugation, and poly A+RNA (mRNA) was purified fromthe total RNA with oligo dT cellurose (Stratagene). Based on thepurified mRNA, rat kidney cDNA library was constructed with cDNA libraryconstructing kit (Stratagene). From the cDNA library, 1,000 genes werepicked up at random, and sequencing was conducted with a vector primer(T3 primer; 5′-AATTAACCCTCACTAAAGGG-3′). As for full-length sequencingof NaGLT1, a primer (SEQ ID NOs: 3 to 12 in the sequence listing) wasdesigned as described in Table 1 mentioned below (custom synthesized byProligo), decoding was conducted by chain terminator method by usingRISA-384 (Shimadzu Corporation), and the sequence listing wasconstructed with GENETYX-MAC Version 10 (SOFTWARE DEVELOPMENT, Tokyo).Actual sequencing was conducted by RISA-384 system (contracting analysisto Shimadzu Corporation, Genomic Research Laboratory). TABLE 1 Locationin SEQ ID Primers Base sequences (5′ → 3′) NaGLT1 NO Forward primers:T3-1 TCGGAAATGGAGTTCCGTGG 105-124 3 T3-2 AGCTGCCTTACTGACTGCCATG 494-5154 T3-3 TACGTATTCTCCTTCGCCACC 996-1016 5 T3-4 TGTGTAACATTGGCAGCCTGG1144-1164 6 T3-5 TAACCCATAGCTGAGGTCTC 1699-1718 7 Reverse primers: T7-1CAGATAGTTGTGAGCCACCATGTG 2095-2072 8 T7-2 GAGTTGCTTAGAGACCTCAGC1728-1708 9 T7-3 AGGTGGTGTACTGCTCAATCC 1293-1273 10 T7-4TCTGAGGCGGCTTCAAAGGATC 757-737 11 T7-5 AAAAGCACCCCACCAACCACAG 409-388 12

Homology analysis was conducted between the obtained gene sequenceinformation and the gene sequences registered in each of data bases,GenBank, EMBL, DDBJ and PDB, by using BLAST, and the information wasclassified into a known group and an unknown group. In regard to about200 kinds of unknown genes, their mRNAs were synthesized in vitro withmCap RNA Capping kit (Stratagene), and a transport experiment wasconducted with the expression system of Xenopus oocytes. As a result, aclone (NaGLT1) which specifically transports metabolism-resistantα-methyl-D-glucopyranoside (αMeGlc) was identified (GenBank accessionNo: AB089802). The isolated NaGLT1 cDNA comprised 2,173 base pairs (SEQID NO: 1) and a protein comprising 484 amino acid residues (SEQ ID NO:2) was encoded in its translation region (111^(th) to 1562^(nd)position) (FIG. 1A). It was presumed that NaGLT1 would be an11-transmembrane glycoprotein according to the result of hydorphobicityanalysis (FIG. 1B).

(Analysis of Distribution of NaGLT1 Expression by Northern Blotting andRT-PCR)

Under the stringent condition [50% formamide, 5×SSPE (composition of1×SSPE: 0.15 M NaCl, 10 mM NaH₂PO₄, and 1 mM EDTA; pH 7.4), 5×Denhardt's solution, 0.2% SDS, and 10 μm/ml DNA derived from herringsperm, at 42° C.], cDNA (full length) of NaGLT1 was labeled with [α-³²P]dCTP (Amersham) by using Prime-a-Gene Labeling System (Promega), andtotal RNA was extracted from each organ of rat with RNeasy mini RNAextraction kit (QIAGEN) and was hybridized to a blotted nylon membrane(Hybond N+ membrane: Amersham). After the hybridization, blottedmembrane was washed twice with 2×SSC (composition of 1×SSC: 0.15 M NaCl,15 mM sodium citrate, pH 7.0)/0.1% SDS for 10 minutes at roomtemperature, and further washed once with 0.5×SSC/0.1% SDS for 30minutes at 42° C. The blotted membrane thus washed was exposed to animaging plate for 3 to 6 hours, visualized bands were read with animaging plate reader, and subsequently, image processing was conductedwith Image Analyze II (Fuji Photo Film Co., Ltd.) (BIO-imaging AnalyzerBAS-2000II system, Fuji Photo Film Co., Ltd.). The value quantificationwas conducted for the intensity of radiation corresponding to each bandon Image Analyze II. As a result, it was observed that NaGLT1 mRNA washighly expressed in kidney cortex and medulla (FIG. 2A).

Then, for the examination by RT-PCR, 1 μg of total RNA was obtained fromeach tissue of rat (brain, heart, lung, liver, small intestine, spleen,kidney cortex, kidney medulla) with RNeasy mini kit (QIAGEN), andreverse transcription reaction was conducted with SuperScript II reversetranscriptase (Invitrogen). After the reaction, remained RNA wasdigested with RNase H (Invitrogen). Heat denaturation was conducted for3 minutes at 95° C. with the obtained single-stranded DNA as a templateby using primers specific to NaGLT1, SGLT1, SGLT2 and GAPDH (see Table 2below and SEQ ID NOs: 13 to 20 in the sequence listing), and Taq DNApolymerase (Takara), then the following cycle was repeated 35 times foramplification. Cycle: heat denaturation for 1 minute at 94° C.,annealing for 1 minute at 58° C., and elongation reaction for 1 minuteat 72° C. RT-PCR product obtained with the cycle was separated on 1.5%agarose gel, and subsequently stained with ethidium bromide andvisualized under UV rays. As a result, it was observed that NaGLT1 washighly expressed in the kidney (cortex, medulla), and other than thekidney, expression over detection limit was observed in the brain, thelung and the liver (FIG. 2B). TABLE 2 SEQ ID Primers Genes (GenBankaccession No.) (Location) NO. NaGLT1 (AB089802) Sense5′-TGGGACCCACATTTCCAGAC-3′ (279-298) 13 Antisense5′-TCTGAGGCGGCTTCAAAGGATC-3′ (736-757) 14 rSGLT1 (D16101) Sense5′-ATGGACAGTAGCACCTTGAGCC-3′ (170-191) 15 Antisense5′-TAGCCCCAGAGAAGATGTCTGC-3′ (647-668) 16 rSGLT2 (U29881) Sense5′-CATTGTCTCAGGCTGGCACTGG-3′ (851-872) 17 Antisense5′-GGACACTGCCACAATGAACACC-3′ (1289-1310) 18 rGAPDH (M17701) Sense5′-CCTTCATTGACCTCAACTAC-3′ (131-150) 19 Antisense5′-GGAAGGCCATGCCAGTGAGC-3′ (705-724) 20(Isolation of Rat Renal Tubule Segments by Microdissection)

Male Wistar rat (7 weeks old) was subjected to midline laparotomy underNembutal anesthesia to expose the left kidney. Cranial side just beforethe diverging point of left renal artery in inferior aorta was ligated.Cranial side just before the lateral diverging point of inferior aortaand inferior vena cava was ligated, and a small hole was made in a bloodvessel at caudal side, immediately under the left renal artery.Subsequently, polyethylene medical tube (PE-50, Becton Dickinson) wasinserted through the small hole to preperfuse the left kidney with 10 mlof solution A (130 mM NaCl, 5 mM KCl, 1 mM NaH₂PO₄, 1 mM magnesiumsulfate, 1 mM calcium lactate, 2 mM sodium acetate, 5.5 mM D-glucose, 10mM HEPES, pH 7.4) by using a 10 ml syringe without putting onoverpressure. After confirming that no congestion, etc., was observed inthe perfused kidney, the kidney was perfused with 10 ml of solution B(solution A containing 1 mg/ml collagenase (type I, Sigma), 1 mg/mlbovine serum albumin (BSA) (Sigma), 10 mM vanadyl-ribonucleoside complex(Invitrogen)), and was quickly removed.

Renal section of 1 to 1.5 mm thick was prepared by cutting the kidney atan angle that covers the cortex and the medulla. The obtained renalsection was oxygenized with 100% O₂ while being shaken in solution B at37° C. for 30 minutes. Then, the renal section was washed with ice-coldsolution A, and each segment of renal tubule mentioned below wasseparated with a siliconized sharp needle while microscope observationwas conducted based on each structural feature. As to each nephronsegment thus isolated (glomerulus, proximal convoluted tubule, proximalstraight tubule, medullary thick ascending limb of Henle, cortical thickascending limb of Henle, cortical collecting duct, outer medullarycollecting duct, inner medullary collecting duct), 20 glomeruli, and 8mm of other segments were used as one sample, and total RNA wasextracted from each segment sample with RNeasy RNA mini kit (QIAGEN).RT-PCR was conducted with the use of the obtained total RNA and theprimer sets (SEQ ID NOs: 21 to 29) in Table 3 below (FIG. 3). As aresult, it has been revealed that NaGLT1 mRNA is highly expressed in theproximal convoluted tubule and the proximal straight tubule, like otherSGLTs (FIG. 3). TABLE 3 Primers Genes (GenBank accession No.) (Location)SEQ ID NO. NaGLT1 (AB089802) Forward primer 5′-CCGGTGTCTCATTTGGTGTTCT-3′(526-547) 21 Reverse primer 5′-ACCCAAGGCGAAACTGAAGTG-3′ (618-638) 22TaqMan probe 5′-ACAAAGGAGCCCCACATATTCAGGCCTT-3′ (589-616) 23 rSGLT1(D16101) Forward primer 5′-CGAGGAGGACCCTAAAGATACCA-3′ (1912-1934) 24Reverse primer 5′-GAACAGGTCATATGCCTTCCTGA-3′ (1977-1999) 25 TaqMan probe5′-TGAAATAGATGCAGAAGCCCCCCAGAAGG-3′ (1936-1964) 26 rSGLT2 (U29881)Forward primer 5′-AAAATACGGCAGGAAGGAACTG-3′ (2117-2138) 27 Reverseprimer 5′-GACAAATTGGCCACCATCTTG-3′ (2193-2213) 28 TagMan probe5′-CCAGTCCATTTGATTGGTTGTCACTTCCC-3′ (2163-2191) 29(Quantitative Analysis of NaGLT1 mRNA Expression Level)

Total RNA derived from the kidney of male Wistar rat (7 weeks old) wasreverse-transcribed (see method 2), with the obtained single-strandedDNA as a template, the expression amounts of NaGLT1, SGLT1, SGLT2 andGAPDH mRNAs were quantitated by using primers specific to NaGLT1, SGLT1,SGLT2 and GAPDH and TaqMan probes (see Table 3) and Universal master mix(Applied Biosystems), and with ABI PRISM 7700 Sequence Detection System.In order to obtain a standard curve, PCR product amplified by real-timePCR was inserted into pGEM-T Easy vector (Promega), and transfected toE. coli (DH-5α). The transfected E. coli was shaking-cultured overnightin LB broth (10 g bactotryptone, 5 g yeast extract, and 10 g NaCl in 1L, pH 7.2), and plasmid DNA encoding amplified product (PCR fragmentsamplified with the primer sets in Table 2) of NaGLT1, SGLT1, SGLT2 andGAPDH, respectively, was purified.

Each concentration was measured with a UV 1200 spectrophotometer(Shimadzu Corporation), and used as control gene at known concentration.Results obtained with PRISM 7700 were numerically converted with astandard curve. The expression amount of GAPDH used as an internalstandard was used for adjusting the amount of template RNA in eachreaction of real-time PCR. As a result, the expression amount of NaGLT1mRNA was indicated to be higher than other SGLTs in both kidney cortexand medulla (FIG. 4).

(Transport Substrate of NaGLT1)

Transportation activity of NaGLT1 was examined with the expressionsystem of Xenopus oocytes (hereinafter abbreviated as oocytes). First,in order to examine the selectivity of various saccharides, NaGLT1 RNAsynthesized in vitro was injected into the oocyte and incubated for 2days at 18° C., then used for the experiment. As a result of examinationof Radio-labeled monosaccharide (α-methyl-D-glucopyranoside (αMeGlc:metabolism-resistant glucose), galactose, fructose, mannose, mannitoland 2-deoxyglucose) and disaccharide (sucrose) as a substrate, uptake ofα-MeGlc only was significantly high in comparison to water-injectedoocytes examined at the same time as a negative control (FIG. 5).Therefore, it was revealed that the transport substrate of NaGLT1 isglucose.

(Property of αMeGlc Transportation Mediated by NaGLT1)

Uptake activity of NaGLT1 cRNA-injected cell and water-injected oocyteto [U-¹⁴C]-α-methyl-D-glucopyranoside (α-MeGlc), D-[1-¹⁴C] galactose,D-[U-¹⁴C] mannose, D-[U-¹⁴C] fructose, [1,2-³H]-2-deoxy-glucose,D-[1-³H] mannitol, [U-¹⁴C] sucrose was measured. At that time,incubation was conducted in a buffer solution comprising 96 mM NaCl, 2mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES (pH 7.4). Further, whenextracellular Na⁺ concentration dependency was conducted, theconcentration of NaCl was adjusted to be within the range of 9.6 to 96mM, and the final osmotic pressure was kept constant by making up forthe shortage to 96 mM with choline chloride (FIGS. 6 and 7).

Next, concentration dependency of αMeGlc uptake was examined by usingNaGLT1-expressing oocytes. As a result, Km value was 3.71±0.09 mM (FIG.6A). In addition, the effect of extracellular Na⁺ ion concentration onthe uptake of αMeGlc mediated by NaGLT1 was analyzed. First,extracellular Na⁺ concentration was varied from 0 to 96 mM, at the statewherein membrane potential difference was eliminated by the addition of7 μM valinomycin, and the effect of extracellular Na⁺ on the uptake ofαMeGlc by rNaGLT1 was examined. Uptake of αMeGlc increased in a mannerdependent to extracellular Na⁺ concentration in cells into which rNaGLT1RNA synthesized in vitro was injected. Further, by examination of dose-or extracellular Na⁺-dependancy to the uptake of αMeGlc by rNaGLT1(FIGS. 6A and 6C), Vmax value (the maximum transport rate) and Km valuewere calculated for each case. Based on those values, Hill plot(logarithmic values of the figure calculated by dividing Vmax bytransport rate V when the abscissa axis is the concentration of variedsubstrate or ion, and ordinate axis is each substrate (or ion)concentration) was conducted (FIGS. 6B and 6D) and Hill coefficient wascalculated. As a result, Hill coefficient of αMeGlc and Na⁺ were 1.06and 1.00, respectively, and coupling ratio of αMeGlc and Na⁺ wasindicated to be 1:1 (FIG. 6). Therefore, it was suggested that NaGLT1 isa glucose transporter which functions in a manner dependent toextracellular Na⁺ ion concentration.

Further, the effect of various inhibitors on the uptake of [¹⁴C]-labeledαMeGlc mediated by NaGLT1-expressing oocytes was examined, and as aresult, unlabeled αMeGlc, D-glucose, 2-deoxyglucose and phloridzin hadan extremely strong inhibitory effect. Fructose and phloretin had a weakinhibitory effect. On the other hand, L-glucose, 3-O-methylglucose,galactose and mannose exhibited no effect on the uptake of αMeGlcmediated by NaGLT1 (FIG. 7).

(Construction of Anti-NaGLT1 Antibody)

Based on the amino acid sequence of NaGLT1, a peptide at C-terminal side(H2N-LPLDRKQEKSINSEGQ-COOH) (SEQ ID NO: 30) was constructed with itsN-terminal side as cysteine (custom synthesized by Sawady TechnologyCo., Ltd.). As a result of analysis with high-performance liquidchromatography (HPCL), the purity of the synthesized peptide was 92%.Then, a conjugate to this peptide was constructed by using hemocyanin(keyhole limpet hemocyanin (Calbiochem-Behring)). The conjugate wasdispensed into 10 containers by 1 ml each, and stored in a frozen state.

As for the conjugate, uniform emulsion was prepared with Freund completeadjuvant (Difco). After collecting preimmunized serum of male Japanesewhite rabbit (2 kg), immunization was conducted at 0.2 mg/rabbit at2-week intervals. Blood was collected at each immunization, and antibodytiter was analyzed by ELISA method. After obtaining sufficient antibodytiter eventually, whole blood was collected and stored in a frozen stateas an anti-serum.

(Localization Analysis of NaGLT1 by Immunoblotting)

Each tissue was extracted from male Wistar rat (220 to 230 g) underpentobarbital anesthesia, and homogenized with a homogenate buffer (230mM sucrose, 5 mM Tris/HCl (pH 7.5), 2 methylenediamine tetra acetic acid(EDTA), 0.1 mM phenylmethylsulfonyl fluoride (PMSF)), and centrifugationwas conducted for 15 minutes at 3000 g. The supernatant was removed andcentrifugation was further conducted for 30 minutes at 24500 g tocollect a precipitate (a crude memberane fraction). Rat renal brushborder membrane and basolateral membrane were prepared simultaneouslyaccording to Percoll density-gradient centrifugation (Biochim BiophysActa 773, 113-124, 1984). The membrane sample was solubilized to SDSsample buffer (2% SDS, 125 mM Tris, 20% glycerol), and polyacrylamideelectrophoresis (Nature 227, 680-685, 1970) was conducted.

As a molecular weight marker, Rainbow™ colored protein molecular weightmarkers [myosin (220,000), phosphorylase beta (97,400), BSA (66,000),ovalbumin (46,000), carbonic anhydrase (30,000), trypsin inhibitor(21,500), lysozyme (14,300)] (Amersham) were used. After theelectrophoresis, the protein was transferred to PVDF membrane (ImmobironP, Millipore), and then rinsed with TBS-T (Tris buffered saline; 20 mMTris/HCl (pH 7.5), 137 mM NaCl, 0.1% Tween 20). Next, after blocking wasconducted with TBS-T containing 5% BSA for 2 hours at room temperature,the membrane was made to react with anti-serum diluted with TBS-Tcontaining 5% BSA (1:2000) at 4° C. overnight, and then the membrane waswashed three times with TBS-T for 15 minutes. By using ECLchemiluminescence kit (Amersham), X-ray film (Fuji Photo Film Co., Ltd.)was exposed. As a result, it was revealed that NaGLT1 protein localizedon renal brush border membrane (FIG. 8). The left part of FIG. 8 showsthe result when the reaction was conducted with an anti-NaGLT1 antibodyonly, and the right part shows the result when the reaction wasconducted with an anti-NaGLT1 antibody which had been treated with anantigen peptide.

(Uptake of Fructose by HEK 293 Cells Mediated by NaGLT1)

As to HEK293 cells incubated with buffer solution containing glucoseanalog (0.1 mM, 37 kBq/ml) for 15 minutes at 37° C., uptake analysis wasconducted 2 days after the transfection of a vector (white column, 0.8μg/well) or rNaGLT1 cDNA (black column, 0.8 μg/well). The results areshown in FIG. 9(A). Each column indicates mean±S.E.M obtained from 3single layers. The value *P<0.05 was significantly different from thatof HEK293 cells transfected with the vector (Student's unpaired t-test).

Uptake of [¹⁴C] fructose by HEK293 cells transfected with rNaGLT1 cDNA(0.8 μg/well) was analyzed in an incubation buffer solution, at variousconcentrations (0.1 to 20 mM), for 15 minutes at 37° C., and the uptakemeasured in HEK293 cells transfected with the vector was deducted. Theresults are shown in FIG. 9(B). The inserted graph shows Eadie-Hofsteeplot of the uptake. Each point indicates mean±S.E.M of 3 single layers.The apparent K_(m) and V_(max) values were obtained from 3 independentexperiments.

(Uptake of Fructose by Renal Brush Border Membrane Vesicles)

Membrane vesicles (20 μl) suspended in 100 mM mannitol and 10 mM HEPES(pH 7.5) were incubated with a substrate mixture (20 μl) containing 100mM mannitol, 200 mM NaCl (black circle: ●) or KCl (white circle: ∘), 4mM [¹⁴C] fructose and 10 mM HEPES (pH 7.5) at 25° C. The results areshown in FIG. 10 (A). The values indicate mean±S.E.M of 3 independentexperiments, respectively. Each experiment was conducted with brushborder membrane vesicles isolated from 5 rats.

Na⁺-dependent uptake of fructose by renal brush border membrane vesicleswas analyzed in the presence of NaCl in an incubation buffer solution ofvarious concentrations (0.1 to 20 mM) for 15 seconds at 25° C., and theuptake in case where NaCl is substituted with KCl was deduced. Theresults are shown in FIG. 10 (B). The inserted graph shows Eadie-Hofsteeplot of the uptake. Each point indicates mean±S.E.M of 3 measurements.The apparent K_(m) and V_(max) values were obtained from 3 independentexperiments.

(Effect of Glucose Analogs, Phloridzin and Phloretin, on the Uptake of[¹⁴C] Fructose by HEK293 Cells Transfected with rNaGLT1 or Renal BrushBorder Membrane Vesicles)

In the presence and absence of phloridzin (50 μM) or phloretin (50 μM),a glucose analog (30 mM), the uptake of [¹⁴C] fructose (0.1 mM, 37kBq/ml) by HEK293 cells transfected with rNaGLT1 was measured for 15minutes at 37° C., and the uptake measured in HEK293 cells transfectedwith a vector was deducted. In the presence and absence of phloridzin(50 μM) or phloretin (50 μM), a glucose analog (30 mM), membranevesicles (20 μl) suspended in 100 mM mannitol and 10 mM HEPES (pH 7.5)were incubated with a substrate mixture (20 μl) containing [¹⁴C]fructose (finally, 2 mM, 74 kBq/ml) for 15 seconds at 25° C., and theuptake in case where NaCl is substituted with KCl was deduced. Theabsence of Na⁺ means the uptake measured under the condition that NaClis substituted with choline chloride. The results are shown in Table 4.The values indicate mean±S.E.M of 3 independent experiments,respectively. The value *P<0.05 was significantly different from that ofcontrol (Fisher's t-test). TABLE 4 Na⁺-dependent uptake Uptake of [¹⁴C]fructose of [¹⁴C] fructose by HEK293 cells by renal brush borderexpressing rNaGLT1 membrane vesicles Pmol/mg % of Pmol/mg % of Treatmentprotein/min control protein/sec control Control 2.92 ± 0.04  100 49.5 ±3.6 100 Absence of Na⁺ 0.16 ± 0.20* 5  −0.2 ± 1.3* 0 Fructose 0.22 ±0.06* 8  5.4 ± 2.1* 11 •MeGlc 1.24 ± 0.13* 42  23.8 ± 2.1* 48 Galactose2.88 ± 0.23  99 35.2 ± 3.3 71 3-OMG 2.60 ± 0.15  89 47.1 ± 4.5 95 2-DG2.04 ± 0.19* 70  16.8 ± 3.7* 34 Sucrose 4.20 ± 0.17* 144 47.3 ± 6.0 962,5-AM 1.16 ± 0.16* 40 38.6 ± 2.6 78 Phloridzin 0.34 ± 0.11* 12  3.2 ±0.8* 6 Phloretin 0.97 ± 0.09* 33 35.8 ± 3.5 72

INDUSTRIAL APPLICABILITY

Renal diabetes is thought to be caused by a defect in renal glucosereabsorption, and the defect in glucose reabsorption is thought to becaused by the deficiency of glucose transporter gene. Because theconventionally unknown gene is obtained by the present invention, itbecomes possible to elucidate, diagnose, prevent/treat renal diabetesand to develop drugs for the disease with the use of the gene and aglucose and/or fructose transporter protein, an expression product ofthe gene.

For example, the isolation of novel genes, diagnosis of glucose and/orfructose transporter function, the detection of genetic diseases in thekidney becomes possible by the isolation of novel glucose and/orfructose transporter genes such as those in the human kidney, and bymeasuring the expression of glucose and/or fructose transporter in humanor rat tissue cells, with the gene and the peptide of the presentinvention, and further, with an antibody that specifically binds to thepeptide.

In addition, it becomes possible to prevent/treat renal genetic diseasesby regulating glucose and/or fructose transporter function in tissuecells by introducing the gene or the antisense strand of the gene of thepresent invention into tissue cells such as renal tissue cells tosuppress the expression of the gene. Moreover, a non-human animal modelfor renal diabetes focused on glucose and/or fructose transporter can beconstructed by constructing an animal wherein the gene of the presentinvention is deficient in its chromosome. By using the non-human animalmodel, the development of novel drugs for preventing/treating renaldiabetes will be possible.

1. A DNA which comprises a base sequence shown by SEQ ID NO: 1 in thesequence listing or its complementary sequence, or a sequence containingpart or whole of these sequences.
 2. A DNA which hybridizes with the DNAaccording to claim 1 under a stringent condition, and which encodes apolypeptide having glucose and/or fructose transporter function.
 3. ADNA which encodes the following polypeptide (a) or (b); (a) apolypeptide which comprises an amino acid sequence shown by SEQ ID NO: 2in the sequence listing, (b) a polypeptide which comprises an amino acidsequence wherein one or a few amino acids are deleted, substituted oradded in the amino acid sequence shown by SEQ ID NO: 2 in the sequencelisting, and which has glucose and/or fructose transporter function. 4.A polypeptide which comprises an amino acid sequence shown by SEQ ID NO:2 in the sequence listing.
 5. A polypeptide which comprises an aminoacid sequence wherein one or a few amino acids are deleted, substitutedor added in the amino acid sequence shown by SEQ ID NO: 2 in thesequence listing, and which has glucose and/or fructose transporterfunction.
 6. A method for producing a polypeptide which has glucoseand/or fructose transporter function, wherein the DNA according toclaims 1 to 3 is incorporated into an expression vector and expressed byintroducing the recombinant expression vector into a host cell.
 7. Anantibody which is induced by using the polypeptide according to claim 4or 5, and which binds to the polypeptide specifically.
 8. The antibodyaccording to claim 7, wherein the antibody is a monoclonal antibody. 9.The antibody according to claim 7, wherein the antibody is a polyclonalantibody.
 10. A method for producing an animal tissue cell expressing apolypeptide which has glucose and/or fructose transporter function,wherein the DNA according to any one of claims 1 to 3 is introduced intoan animal tissue cell.
 11. The method for producing an animal tissuecell expressing a polypeptide which has glucose and/or fructosetransporter function according to claim 10, wherein the animal tissuecell is a tissue cell of rat kidney, an epithelial cell derived fromporcine kidney, an epithelial cell derived from canine kidney or anepithelial cell derived from opossum kidney.
 12. The method forproducing an animal tissue cell expressing a polypeptide which hasglucose and/or fructose transporter function according to claim 10,wherein the animal tissue cell is HEK293, a transfected human embryonickidney cell line.
 13. An animal tissue cell expressing a polypeptidewhich has glucose and/or fructose transporter function, which isproduced by the method according to any one of claims 10 to
 12. 14. Amethod for screening a substance having a glucose and/or fructosetransporter function-regulating activity, wherein an effect of a testsubstance on glucose transport function is measured with the use of theanimal tissue cell expressing a polypeptide which has glucose and/orfructose transporter function according to claim
 13. 15. A non-humananimal model which develops renal diabetes caused by a defect in renalglucose reabsorption, whose gene function to express a polypeptide whichhas glucose and/or fructose transporter function shown by SEQ ID NO: 2in the sequence listing is deficient in its chromosome.
 16. Thenon-human animal model which develops renal diabetes according to claim15, wherein the deficiency in the gene function to express a polypeptidewhich has glucose and/or fructose transporter function is deficiency ina function of a gene which expresses a polypeptide which has glucoseand/or fructose transporter function shown by SEQ ID NO: 1 in thesequence listing.
 17. A method for screening a preventive/therapeuticdrug for renal diabetes caused by a defect in glucose reabsorption,wherein a test substance is administered to the non-human animal modelwhich develops renal diabetes caused by a defect in renal glucose and/orfructose reabsorption according to claim 15 or 16, and glucosereabsorption ability of the non-human animal model, or a cell, a tissueor an organ of the non-human animal model is measured/evaluated.
 18. Aprobe for diagnosing glucose and/or fructose transporter functioncomprising whole or part of an antisense strand of the base sequenceaccording to claim
 1. 19. A microarray or a DNA chip for diagnosingglucose and/or fructose transporter function, wherein at least one DNAaccording to any one of claims 1 to 3 is immobilized.
 20. Apharmaceutical for diagnosing glucose and/or fructose transporterfunction, wherein the antibody according to any one of claims 7 to 9and/or the probe for diagnosing according to claim 18 is prepared.
 21. Amethod for diagnosing glucose and/or fructose transporter function,wherein a sample is obtained from a test substance, and the expressionof the gene according to claim 1 in the sample is measured.
 22. A methodfor diagnosing glucose and/or fructose transporter function, wherein themeasurement of the gene expression according to claim 21 is conductedwith the probe for diagnosing glucose and/or fructose transporterfunction according to claim 18, or with the microarray or the DNA chipfor diagnosing glucose and/or fructose transporter function according toclaim
 19. 23. A method for diagnosing glucose and/or fructosetransporter function, wherein a sample is obtained from a test substanceand cultured, and the polypeptide according to claim 4 produced by theexpression of the gene in the sample is measured.
 24. A method fordiagnosing glucose and/or fructose transporter function, wherein themeasurement of the polypeptide according to claim 23 is conducted withthe antibody according to any one of claims 7 to
 9. 25. A method fordiagnosing a renal disease, wherein the diagnosis of glucose and/orfructose transporter function according to any one of claims 21 to 24 ismeasurement of glucose and/or fructose transporter function in a renaldisease.
 26. A method for regulating glucose and/or fructose transporterfunction in an animal tissue cell, wherein the DNA according to any oneof claims 1 to 3 is introduced into an animal tissue cell.
 27. A methodfor regulating glucose and/or fructose transporter function in an animaltissue cell, wherein the expression of the DNA according to claim 1 issuppressed in an animal tissue cell.
 28. A method for regulating glucoseand/or fructose transporter function in an animal tissue cell, whereinthe expression of the DNA according to claim 1 is suppressed in ananimal tissue cell by introducing whole or part of an antisense strandof the DNA base sequence according to claim 1 into an animal tissuecell.
 29. The method for regulating glucose and/or fructose transporterfunction in an animal tissue cell according to any one of claims 26 to28, wherein the animal tissue cell is an animal kidney cell.